Theses & Dissertations

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    Anabaena Sensory Rhodopsin Reconstituted in Nanodiscs as a Promising Platform for Structural Studies
    Camargo, Suelen; Brown , Leonid
    Membrane protein studies need an excellent membrane-mimicking reconstitution system. Nanodiscs offer many advantages in this regard but require optimal conditions for achieving homogeneous and stable samples. Model protein Anabaena Sensory Rhodopsin (ASR) characterization in nanodiscs provides insights into protocol steps of membrane proteins reconstitution. Size-exclusion chromatography and electron microscopy aided in sample evaluation, identifying aggregation and the disk diameter, making possible estimation of ASR oligomeric state. Solution-state NMR showed ASR stability in nanodiscs for at least five days. Comparing liposomes and nanodiscs via ssNMR showed that ASR structure is mostly unaffected, minimally impacting loops. Helical arrangement alters somewhat, due to lipid composition and MSP scaffold influence, possibly causing direct MSP-ASR interaction. ssNMR detected co-purified PE lipids but not ECAPG, suggesting that ASR in nanodiscs might lack these sugar moieties due to structural changes. This research contributes to an improved comprehension of ASR's structure and oligomerization behavior across varied reconstitution platforms.
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    Investigating the chromophore and binding pocket structure of a novel inward proton pump, GSS AntR, using biosynthetic 13C-retinal
    (University of Guelph, ) Pinto, Marie; Brown, Leonid
    Microbial rhodopsins are light-activated proteins with diverse physiological roles across all domains of life. A new member of the schizorhodopsin family, Antarctic rhodopsin (AntR) was found in freshwater lakes and acts as an inward proton pump. Its structure and biophysical properties are established, but its mechanism of action remains elusive. We optimized the biosynthetic production of 13C-all-trans-retinal for investigation of an AntR homolog with a GSS motif on helix C using Magic Angle Spinning solid-state NMR spectroscopy. Chemical shift values for all twenty of retinal’s carbon atoms were assigned and compared with bacteriorhodopsin, an extensively studied outward proton pump. Raman spectroscopy and molecular dynamics simulations confirmed that retinal adopts a unique conformation due to electrostatic interactions with neighbouring residues. Understanding activation mechanisms elucidates precise evolutionary steps taken to develop unconventional protein functions necessary for host survival and how they may advance optogenetic progress.
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    Pairing in nuclear and cold atomic systems
    (University of Guelph, ) Palkanoglou, Georgios; Gezerlis, Alexandros
    Pairing correlations in nuclear systems have a long and rich history. They are an effect suggested more that a half-century ago and they are still related to a sizeable part of theoretical and experimental nuclear structure and dynamics investigations, while their relevance to the properties of neutron stars make nuclear superfluids an important concept in neutron star structure studies. In this thesis, we present various phenomenological investigation of nuclear superfluidity. We prescribe a modern way of solving the mean-field equations of pairing, holding the promise of out-performing standard approaches. On a different front, we complement novel microscopic descriptions of neutron pairing by extracting error estimations in systematic ways. Finally, we are probing novel nuclear superfluids, such as spin-triplet or mixed-spin pairs in nucleon-“emulating” cold atoms and in nuclei. For the latter, we identify the behavior of various pairing condensates under realistic nuclear deformation, bringing results closer to experiment.
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    Dynamical self-consistent field theory simulation of high-generation, dendritic phytoglycogen nanoparticles
    (University of Guelph, ) Morling, Benjamin; Dutcher, John; Wickham, Robert
    Phytoglycogen (PG) is a naturally occurring, glucose dendrimer that is extracted from sweet corn as compact, 22 nm radius nanoparticles. Extensive experimental studies have been performed to characterize the structural and hydration properties of PG; however, little work has been done to develop a realistic model of PG. The work in this thesis is dedicated to the development of an efficient model of a PG nanoparticle solubilized in water using dynamical self-consistent field theory. We improve the efficiency of our model by exploiting the dendritic architecture of PG to decompose the bead-spring dynamics of the entire dendrimer into the independent dynamics of its constituent sub-chains. By varying the strength of the interactions between PG and water, we are able to tune the morphology, size, and hydration of the nanoparticle to be in agreement with previous small-angle neutron scattering, rheology, and atomic force microscopy measurements.
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    A Membrane Photosensor Related to Proteorhodopsin with Unique Motifs for Signal Transduction
    (University of Guelph, ) Saliminasab, Maryam; Brown, Leonid
    Microbial rhodopsins are light-activated retinal-binding membrane proteins, performing a variety of ion transporting and photosensory functions in prokaryotic and eukaryotic cells. They display several cases of convergent evolution, where the same function is produced by unrelated or very distant protein groups. For example, both schizorhodopsins and xenorhodopsins are inward proton pumps, while halorhodopsins and NTQ-rhodopsins are inward chloride pumps. Here we present another possible case of such convergent evolution, describing biophysical properties of a new group of sensory rhodopsins, not related to the well-known haloarchaeal ones. The first representative of this group was identified in 2004 (by Kyndt, Meyer, and Cusanovich) but none of the members had been expressed and characterized. The well-studied haloarchaeal sensory rhodopsins interacting with membrane-embedded methyl-accepting Htr transducers are close relatives of halobacterial proton pump bacteriorhodopsin and have been studied extensively. In contrast, the new group of sensory rhodopsins we describe here is a relative of proteobacterial proton pumps, proteorhodopsins, but appear to interact with Htr-like transducers likewise. This interaction is likely to occur through an unknown mechanism, as they do not conserve the residues found important for interaction of haloarchaeal sensory rhodopsins and their cognate transducers. Moreover, the new sensory rhodopsins have unique structural motifs and many unusual amino acid residues, including those around the retinal chromophore, most strikingly, a tyrosine in place of a carboxyl counterion of the retinal Schiff base on helix C. We describe spectroscopic properties and molecular dynamics simulations of these sensory rhodopsins, which report on their unique structure and hydrogen-bonded networks, their unusual retinal chromophore, and probe their interactions with the transducers. To characterize their unique sequence motifs, we augment the spectroscopy and biochemistry data by structural modeling of the wild type and three mutants. Taken together, the experimental data, bioinformatics sequence analyses, and structural modeling suggest that the tyrosine/aspartate complex counterion contributes to a complex water-mediated hydrogen-bond network that couples the protonated retinal Schiff base to an extracellular carboxylic dyad.